Formation of toxic 2-nonyl-p-benzoquinones from α-tertiary 4-nonylphenol isomers during microbial metabolism of technical nonylphenol.

In many environmental compartments, microbial degradation of α-quaternary nonylphenols proceeds along an ipso-substitution pathway. It has been reported that technical nonylphenol contains, besides α-quaternary nonylphenols, minor amounts of various α-H, α-methyl substituted tertiary isomers. Here, we show that potentially toxic metabolites of such minor components are formed during ipso-degradation of technical nonylphenol by Sphingobium xenophagum Bayram, a strain isolated from activated sewage sludge. Small but significant amounts of nonylphenols were converted to the corresponding nonylhydroquinones, which in the presence of air oxygen oxidized to the corresponding nonyl-p-benzoquinones-yielding a complex mixture of potentially toxic metabolites. Through reduction with ascorbic acid and subsequent analysis by gas chromatography-mass spectrometry, we were able to characterize this unique metabolic fingerprint and to show that its components originated for the most part from α-tertiary nonylphenol isomers. Furthermore, our results indicate that the metabolites mixture also contained several α, ß-dehydrogenated derivatives of nonyl-p-benzoquinones that originated by hydroxylation induced rearrangement, and subsequent ring and side chain oxidation from α-tertiary nonylphenol isomers. We predict that in nonylphenol polluted natural systems, in which microbial ipso-degradation is prominent, 2-alkylquinone metabolites will be produced and will contribute to the overall toxicity of the remaining material.

An unexpected gene cluster for downstream degradation of alkylphenols in Sphingomonas sp. strain TTNP3.

In silico analysis of nucleotide sequences flanking the recently found hydroquinone dioxygenase in Sphingomonas sp. strain TTNP3 revealed a gene cluster that encodes a hydroquinone catabolic pathway. In addition to the two open-reading frames encoding the recently characterized hydroquinone dioxygenase, the cluster consisted of six open-reading frames. We were able to express the three open-reading frames, hqdC, hqdD, and hqdE, and demonstrated that the three gene products, HqdC, HqdD, and HqdE had 4-hydroxymuconic semialdehyde dehydrogenase, maleylacetate reductase, and intradiol dioxygenase activity, respectively. Surprisingly, the gene cluster showed similarities to functionally related clusters found in members of the ß- and γ-proteobacteria rather than to those found in other members of the genus Sphingomonas sensu latu.

ipso-substitution: a general biochemical and biodegradation mechanism to cleave alpha-quaternary alkylphenols and bisphenol A.

Sphingobium xenophagum Bayram is capable of metabolizing 4-alkoxyphenols and endocrine disruptive alpha-quaternary 4-nonylphenols by an ipso-substitution mechanism that involves ring hydroxylation at the site of the substituent. Here, we show that Bayram's ipso-hydroxylating activity was able to transform also bisphenol A (= dimethyl-4,4'-methylenediphenol; BPA) and 4-isopropylphenol. We identified six metabolites when resting cells of strain Bayram were incubated with BPA. They were unambiguously characterized by HPLC-UV, HPLC/MS, and HPLC/MS/MS as hydroquinone, 2-(4-hydroxyphenyl)isopropanol, 4-isopropenylphenol, 4-isopropylphenol, 4-hydroxy-4-isopropenylcyclohexa-2,5-dien-1-one, and 4-hydroxy-4-isopropylcyclohexa-2,5-dien-1-one. In experiments with 4-isopropylphenol as a substrate, 4-hydroxy-4-isopropylcyclohexa-2,5-dien-1-one, one of the metabolites from BPA, accumulated to a high degree. We could rationalize the formation of all metabolites by invoking ipso-hydroxylation and ipso-substitution mechanisms. On closer view, also classical bacterial metabolism of BPA can be well rationalized by an ipso-substitution mechanism, albeit with ipso-attack of an internal alkyl radical instead of an activated oxygen species. This highlights the important role of ipso-substitution as a versatile degradative principle utilized by diverse organisms to degrade alpha-quaternary 4-nonylphenols, 4-alkoxyphenols, and BPA.

Environmental fate of phenolic endocrine disruptors: field and laboratory studies.

Alkylphenolic compounds derived from microbial degradation of non-ionic surfactants became a major focus of environmental research in the early 1980s. More toxic than the parent compounds and weakly oestrogenic, certain metabolites of nonylphenol polyethoxylate (NPnEO) surfactants, especially nonylphenol (NP), raised sustained concern over the risk they pose to the environment and triggered legal measures as well as partly voluntary actions by the manufacturing industry. Continuous progress in the development of analytical techniques is crucial to understand how these alkylphenolic compounds behave in wastewater treatment, the aquatic environment and in laboratory experiments. Measured concentrations and mass flows of phenolic endocrine disruptors, particularly nonylphenolic compounds, bisphenol A and parabens in municipal wastewater effluents and in the Glatt River, Switzerland, show that rain events leading to discharges of untreated wastewater into rivers have a great impact on the riverine mass flows of contaminants. Biotransformation experiments in our laboratory with nonylphenoxyacetic acid and individual NP isomers enabled the elucidation of degradation pathways of these compounds. The finding that nonylphenoxyacetic acid is metabolized via NP further underscores the role of NP as the most relevant metabolite in the degradation of NPnEO. Several Sphingomonadaceae bacterial strains were found to degrade alpha-quaternary 4-NP isomers by an ipso-substitution mechanism, and to use only the aromatic part of the molecule. These reactions turned out to be isomer specific, meaning that rate and extent of transformation depend on constitution, and possibly also on the absolute configuration of the alkyl side chain of a specific isomer. The observation that NP isomers with distinct oestrogenic activities are differentially degraded has significant implications for risk assessment.

Isomer-specific determination of 4-nonylphenols using comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry.

Technical nonylphenol (tNP), used for industrial production of nonylphenol polyethoxylate surfactants, is a complex mixture of C(3-10)-phenols. The major components, 4-nonylphenols, are weak endocrine disruptors whose estrogenicities vary according to the structure of the branched nonyl group. Thus, accurate risk assessment requires isomer-specific determination of 4-NPs. Comprehensive two-dimensional gas chromatography/time-of-flight mass spectrometry (GC x GC/ToFMS) was used to characterize tNP samples obtained from seven commercial suppliers. Under optimal chromatographic conditions, 153-204 alkylphenol peaks, 59-66 of which were identified as 4-NPs, were detected. The 4-NPs comprised approximately 86-94% of tNP, with 2-NPs and decylphenols making up approximately 2-9% and approximately 2-5%, respectively. The tNP products were analyzed for eight synthetic 4-NP isomers, and results were compared with published data based on GC/MS analysis. Significant differences were found among the products and between two samples from a single supplier. The enhanced resolution of GC x GC coupled with fast mass spectral data acquisition by ToFMS facilitated identification of all major 4-NP isomers and a number of previously unrecognized components. Analysis of tNP altered by the bacterium, Sphingobium xenophagum Bayram, revealed several persistent 4-NPs whose structures and estrogenicities are presently unknown. The potential of this technology for isomer-specific determination of 4-NP isomers in environmental matrices is demonstrated using samples of wastewater-contaminated groundwater and municipal wastewater.

Isomer-specific degradation and endocrine disrupting activity of nonylphenols.

Degradation of technical nonylphenol by Sphingobium xenophagum Bayram led to a significant shift in the isomers composition of the mixture. By means of gas chromatography-mass spectrometry, we could observe a strong correlation between transformation of individual isomers and their alpha-substitution pattern, as expressed by their assignment to one of six mass spectrometric groups. As a rule, isomers with less bulkiness at the alpha-carbon and those with an optimally sized main alkyl chain (4-6 carbon atoms) were degraded more efficiently. By mass spectrometric analysis, we identified the two most recalcitrant main isomers of the technical mixture (Group 4) as 4-(1,2-dimethyl-1-propylbutyl)phenols (NP193a and NP193b), which are diastereomers with a bulky alpha-CH3, alpha-CH(CH3)C2H5 substitution. Our experiments with strain Bayram show that the selective enrichment of isomers with bulky alpha-substitutions observed in nonylphenol fingerprints of natural systems can be caused by microbial ipso-hydroxylation. Based on the yeast estrogen assay (YES), we established an estrogenicity ranking with a variety of single isomers and compared it to rankings obtained with different reporter cell systems. Structure-activity relationships derived from these data suggest that Group 4 isomers have a high estrogenic potency. This indicates a substantial risk that enrichment of highly estrogenic isomers during microbial degradation by ipso-substitution will increase the specific estrogenicity of aging material.

Elucidation of the ipso-substitution mechanism for side-chain cleavage of alpha-quaternary 4-nonylphenols and 4-t-butoxyphenol in Sphingobium xenophagum Bayram.

Recently we showed that degradation of several nonylphenol isomers with alpha-quaternary carbon atoms is initiated by ipso-hydroxylation in Sphingobium xenophagum Bayram (F. L. P. Gabriel, A. Heidlberger, D. Rentsch, W. Giger, K. Guenther, and H.-P. E. Kohler, J. Biol. Chem. 280:15526-15533, 2005). Here, we demonstrate with 18O-labeling experiments that the ipso-hydroxy group was derived from molecular oxygen and that, in the major pathway for cleavage of the alkyl moiety, the resulting nonanol metabolite contained an oxygen atom originating from water and not from the ipso-hydroxy group, as was previously assumed. Our results clearly show that the alkyl cation derived from the alpha-quaternary nonylphenol 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol through ipso-hydroxylation and subsequent dissociation of the 4-alkyl-4-hydroxy-cyclohexadienone intermediate preferentially combines with a molecule of water to yield the corresponding alcohol and hydroquinone. However, the metabolism of certain alpha,alpha-dimethyl-substituted nonylphenols appears to also involve a reaction of the cation with the ipso-hydroxy group to form the corresponding 4-alkoxyphenols. Growth, oxygen uptake, and 18O-labeling experiments clearly indicate that strain Bayram metabolized 4-t-butoxyphenol by ipso-hydroxylation to a hemiketal followed by spontaneous dissociation to the corresponding alcohol and p-quinone. Hydroquinone effected high oxygen uptake in assays with induced resting cells as well as in assays with cell extracts. This further corroborates the role of hydroquinone as the ring cleavage intermediate during degradation of 4-nonylphenols and 4-alkoxyphenols.

Differential degradation of nonylphenol isomers by Sphingomonas xenophaga Bayram.

Sphingomonas xenophaga Bayram, isolated from the activated sludge of a municipal wastewater treatment plant, was able to utilize 4-(1-ethyl-1,4-dimethylpentyl)phenol, one of the main isomers of technical nonylphenol mixtures, as a sole carbon and energy source. The isolate degraded 1 mg of 4-(1-ethyl-1,4-dimethylpentyl)phenol/ml in minimal medium within 1 week. Growth experiments with five nonylphenol isomers showed that the three isomers with quaternary benzylic carbon atoms [(1,1,2,4-tetramethylpentyl)phenol, 4-(1-ethyl-1,4-dimethylpentyl)phenol, and 4-(1,1-dimethylheptyl)phenol] served as growth substrates, whereas the isomers containing one or two hydrogen atoms in the benzylic position [4-(1-methyloctyl)phenol and 4-n-nonylphenol] did not. However, when the isomers were incubated as a mixture, all were degraded to a certain degree. Differential degradation was clearly evident, as isomers with more highly branched alkyl side chains were degraded much faster than the others. Furthermore, the C9 alcohols 2,3,5-trimethylhexan-2-ol, 3,6-dimethylheptan-3-ol, and 2-methyloctan-2-ol, derived from the three nonylphenol isomers with quaternary benzylic carbon atoms, were detected in the culture fluid by gas chromatography-mass spectrometry, but no analogous metabolites could be found originating from 4-(1-methyloctyl)phenol and 4-n-nonylphenol. We propose that 4-(1-methyloctyl)phenol and 4-n-nonylphenol were cometabolically transformed in the growth experiments with the mixture but that, unlike the other isomers, they did not participate in the reactions leading to the detachment of the alkyl moiety. This hypothesis was corroborated by the observed accumulation in the culture fluid of an as yet unidentified metabolite derived from 4-(1-methyloctyl)phenol.

A novel metabolic pathway for degradation of 4-nonylphenol environmental contaminants by Sphingomonas xenophaga Bayram: ipso-hydroxylation and intramolecular rearrangement.

Several nonylphenol isomers with alpha-quaternary carbon atoms serve as growth substrates for Sphingomonas xenophaga Bayram, whereas isomers containing hydrogen atoms at the alpha-carbon do not. Three metabolites of 4-(1-methyloctyl)-phenol were isolated in mg quantities from cultures of strain Bayram supplemented with the growth substrate isomer 4-(1-ethyl-1,4-dimethyl-pentyl)-phenol. They were unequivocally identified as 4-hydroxy-4-(1-methyl-octyl)-cyclohexa-2,5-dienone, 4-hydroxy-4-(1-methyl-octyl)-cyclohex-2-enone, and 2-(1-methyl-octyl)-benzene-1,4-diol by high pressure liquid chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy. Furthermore, two metabolites originating from 4-n-nonylphenol were identified as 4-hydroxy-4-nonyl-cyclohexa-2,5-dienone and 4-hydroxy-4-nonyl-cyclohex-2-enone by high pressure liquid chromatography-mass spectrometry. We conclude that nonylphenols were initially hydroxylated at the ipso-position forming 4-alkyl-4-hydroxy-cyclohexa-2,5-dienones. Dienones originating from growth substrate nonylphenol isomers underwent a rearrangement that involved a 1,2-C,O shift of the alkyl moiety as a cation to the oxygen atom of the geminal hydroxy group yielding 4-alkoxyphenols, from which the alkyl moieties can be easily detached as alcohols by known mechanisms. Dienones originating from nongrowth substrates did not undergo such a rearrangement because the missing alkyl substituents at the alpha-carbon atom prevented stabilization of the putative alpha-carbocation. Instead they accumulated and subsequently underwent side reactions, such as 1,2-C,C shifts and dihydrogenations. The ipso-hydroxylation and the proposed 1,2-C,O shift constitute key steps in a novel pathway that enables bacteria to detach alpha-branched alkyl moieties of alkylphenols for utilization of the aromatic part as a carbon and energy source.